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Students
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Cheryl
Gomillion
Biosystems Engineering
Applied Biotechnology Concentration
Summer
Research
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During the
summer of 2002 I participated in a ten week Research Experiences
for Undergraduates (REU) program sponsored by the National Science
Foundation and held at Clemson University. I completed a research
project in the Tissue Engineering Laboratory of the Bioengineering
Department. My project research is described as follows.
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Cell
Adhesion and Tissue Engineered Vessels
Advisors: Dr. Karen Burg, David Orr, Chuck Thomas
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Background
Blood vessels in our bodies are used to transport blood to and
from our organs. There are three types: Arteries (carry blood
away from heart), Veins (carry blood to the heart), and Capillaries
(connect arteries to veins). The basic structure of blood vessels
is a tube of three layers, the intima, media, and adventitia.
The outermost layer of the adventitia serves as structural support
for the vessel, providing its shape and rigidity. It is composed
of a fibrous material and smooth muscle cells (SMC) The central
layer of the media is comprised of SMC and fibers of elastic tissue.
The innermost layer, the intima, is composed of connective tissue
and a lining of endothelial cells (EC) adjacent to blood flow.
The primary components of the vessels, though, are endothelial
cells because they help to prevent thrombus formation by releasing
anticoagulants, and smooth muscle cells because they are elastic
by nature and allow for expansion and contraction by the vessel
as blood is pumped throughout the body.
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Normal
Vessel Anatomy
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Project Motivation
Atherosclerosis, hardening and narrowing of the arteries due to
plaque formation, accounts for nearly 75% of all deaths from cardiovascular
disease.1 Current treatments for atherosclerosis include medications
(anti-platelets and cholesterol-reducing drugs), catheter based
procedures (balloon angioplasty, stents, or atherectomy), and
vascular grafts which are used in more severe cases where the
actual vessel or artery has to be replaced. There are two categories
of vascular grafts: biological (Autografts-long saphenous vein
from calf of patient and Allografts-vein from other source of
same species) and synthetic (Man-made vessels fabricated from
Dacron®, Teflon®, or polyurethane). Current complications
that exist with the use of the synthetic grafts include inability
to obtain small-diameter vessels (inner diameter <5mm), thrombus
formation, graft occlusion, graft infection, distal embolisms,
and lack of endothelial lining.
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Blood
Clot
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To eliminate
the need for using synthetic materials in the body, researchers
have become more involved in an emerging field of bioengineering
research called tissue engineering.Tissue engineering is the development
and manipulation of laboratory-grown molecules, cells, tissues,
or organs to replace or support the function of defective or injured
body parts. |
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scaffold made of an absorbable polymer is constructed in the shape
of the desired structure. The scaffold is then seeded with cells
on the surface in vitro, and grown in a media supplemented with
growth factors. The structure is implanted into the patient and
the cells are allowed to grow and attach to surrounding tissues.The
use of tissue engineering will allow for the development of fully
biological vessel substitutes that mimic the performance of natural
vessels, reducing the risks involved. |
Tissue Engineered Blood Vessels
Previous research with tissue engineered vascular grafts has led
to the development of 3 types of engineered grafts. The first
type utilizes a tube shaped biodegradable scaffold that would
be made of one of the absorbable polymers. Cells would be seeded
onto the scaffold and implanted into the body. The use of cellular
sheets was done by Nicolas L’heureux and his group of researchers
in Quebec. They constructed an entirely cellular vessel in vitro
using a sheet of fibroblast cells to form the adventitia (outer
layer), a sheet of smooth muscle cells to form the media, and
a sheet of endothelial cells to form the intima. This photo shows
one of the vessels, as it was developed, and how they can vary
in size.
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View
of a mature TEBV (9 weeks of adventitial maturation)3.
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of the primary problems with this method, as with other methods
is the lack of structural integrity, especially once pressure
is created by blood flow when implanted into the body. Another
major problem that exists is the detachment of the endothelium
cell lining from the vessel surface once blood flow is introduced.
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Summer
Research: Cell Adhesion Studies
In order to better develop tissue engineered vessels that do not
have the problem of cellular detachment, my research was focused
on evaluating the cell adhesion properties of the SMC and EC to
various materials and comparing their adhesiveness to the materials.
Factors that could potentially influence the adhesive properties
of the cell types include the substrate material (the type of
material the cells are grown on), the seeding density (the amount
of cells initially placed in the culture to grow), and the origin
of the cells. Various studies were performed first using fibroblast
cells to determine the binding characteristics of the cells to
the different film types, in relation to the cell concentration
and flow rates. Once cell growth was actually observed on the
surface of the films, the circular films were placed in a flow
chamber for a specific time interval and flow rate, after which
they were evaluated under an inverted microscope to observe the
amount of cells retained after being subjected to flow. Conditions
were varied during the fibroblast studies to determine which conditions
provided the largest retention of a measurable quantity after
being subjected to flow. Once the ideal conditions and parameters
were determined using the fibroblast cells, the SMC and EC were
grown under these same conditions and then tested in the flow
chamber. After being subject to the flow, the films were evaluated
under the microscope and conclusions regarding the cellular retention
could be drawn.
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Cell
Passage: 29
Seeding Time: 72 hours
Cell Quantity: 6x104
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Flow
Rate: 150ml/min
Cell Coverage: 90-95%
Flow Duration: 5 min
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Film
Materials Used to Grow Cells On
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Flow
Chamber Used to Test Cell Adhesion
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